Eukaryotic cell compartmentalization requires the targeting of various components, e.g. proteins, lipids, etc, into specific subcellular structures. In humans, mistargeting often results in serious disease. Targeting of proteins invariably involves their transport across or into membranes. At present, the mechanisms by which this is accomplished are largely unknown. Our long term goal is to understand biochemical mechanisms involved in the assembly of cell membranes, especially the protein components of those membranes. The thylakoid membrane within plant chloroplasts has been chosen as a model to achieve this goal. Nuclear-encoded thylakoid membrane proteins are localized in a three-stage process that involves: transport across the two envelope membranes, protein-mediated traversal of the stroma, and insertion into the thylakoid bilayer. In vitro reconstituted assays for each step of this process have been developed. This proposal describes biochemical studies designed ultimately to determine underlying mechanisms of each step. Analysis of changes in envelope proteins during a light-mediated activation of the protein import apparatus will identify protein components of the translocation machinery. The stromal protein factor(s) required to maintain membrane protein solubility and insertion competence will be purified and its mode of action determined. Purification will be accomplished by conventional as well as affinity techniques. Finally, the steps of thylakoid protein insertion and assembly will be determined. This will be accomplished by withholding or lowering the requirements for the process and characterizing intermediates. The ability to arrest intermediates should provide a means of identifying protein components of the insertion apparatus. Successful completion of this project will provide fundamental insight into the manner by which proteins can differentially interact with two protein translocation systems and will elucidate the manner by which soluble protein(s) within organelles can stabilize membrane proteins prior to their insertion into the bilayer.